The alarming world-wide increase in multidrug-resistant and extensively drug-resistant tuberculosis calls for new antimycobacterial drugs against new types of targets that are less likely to undergo mutations that lead to drug-resistance. The discovery that three genes of M. tuberculosis, recA, dnaB and sufB, are interrupted by inteins, which must be excised from inactive precursor proteins by the process of protein splicing to yield functional proteins, suggests protein splicing as a novel target for anti-TB drugs. Since two of these genes (dnaB and sufB) are essential for growth and the third (recA) predisposes to drug-resistance mutations, protein splicing inhibitors, which disable two separate vital functions and at the same time suppress mutation rates, would be unlikely to elicit drug-resistance. A robust, protein- based high-throughput screening system for protein splicing inhibitors has been developed and used for screening commercial small-molecule compound libraries, leading to nearly 60 confirmed positives with IC50 values in the low micromolar range. Most of the confirmed protein splicing inhibitors were electrophiles that inhibit protein splicing irreversibly by binding to its catalytic cysteine, but a few non-covalent inhibitors were also found. The same assay will be used to screen the MLPCN library, and the hits obtained will be reviewed for compound classes based on the likelihood of covalent modification. The hits will then be re-screened in the presence of a large excess of a thiol, which is known to abrogate the inhibition of protein splicing by the thiol-reactive compounds in order to discriminate between covalent and non- covalent inhibitors. Confirmed positives of both inhibitor classes will be screened for the inhibition of the growth of virulent strains of M. tuberculosis and for cytotoxicity using animal cells to identify candidates for lead optimization and further testing as antimycobacterial drugs. PUBLIC HEALTH RELEVANCE: Statement Tuberculosis (TB) is globally the most widespread infectious disease. Two billion people, one- third of the world's population, are infected with Mycobacterium tuberculosis and 5-10% of these suffer active disease, leading to nearly 3 million deaths annually. The alarming world-wide increase in multidrug-resistant and extensively drug-resistant TB calls for new antimycobacterial drugs against new types of targets that are less likely to undergo mutations that lead to drug- resistance. This proposal seeks to identify inhibitors of protein splicing, which is essential for the expression of three genes in M. tuberculosis. Two of these genes are essential for growth and the third predisposes to drug-resistance mutations. Protein splicing inhibitors, by disabling two separate vital functions and at the same time suppressing mutation rates, should therefore be highly effective anti-TB drugs that would be unlikely to elicit drug-resistance.